Comparison of Real-Time PCR Detection Chemistries and Cycling Modes Using Mon810 Event-Specific Assays as Model

The most widely accepted methods for accurate quantitative detection of genetically modified organisms rely on real-time PCR. Various detection chemistries are available for real-time PCR. They include sequence-unspecific DNA labeling dyes such SYBR-Green I and the use of both universal (e.g., Ampli...

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Bibliographic Details
Published in:Journal of agricultural and food chemistry 2007-05, Vol.55 (11), p.4312-4318
Main Authors: La Paz, José Luis, Esteve, Teresa, Pla, Maria
Format: Article
Language:English
Subjects:
Biological and medical sciences
corn
detection
DNA probes
DNA, Plant - analysis
economic costs
fluorescence
fluorescent labeling
food crops
Food industries
foods
Fundamental and applied biological sciences. Psychology
General aspects
genetically modified foods
green fluorescent protein
hydrolysis
Methods of analysis, processing and quality control, regulation, standards
nucleic acid hybridization
Plants, Genetically Modified - chemistry
Polymerase Chain Reaction - methods
reliability
reverse transcriptase polymerase chain reaction
sequence analysis
transgenic plants
validity
Zea mays
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Summary:The most widely accepted methods for accurate quantitative detection of genetically modified organisms rely on real-time PCR. Various detection chemistries are available for real-time PCR. They include sequence-unspecific DNA labeling dyes such SYBR-Green I and the use of both universal (e.g., AmpliFluor) and sequence-specific double-labeled probes, the latter comprising hybridization (e.g., Molecular Beacon) and hydrolysis (e.g., TaqMan or MGB) probes. Also, new real-time PCR devices and reagents allowing fast cycling reactions exist. Five Mon810 real-time PCR assays were developed in which the event specificity was based on the detection of transgene and plant rearranged sequences found to 3‘ flank the insertion site. Every assay was specifically designed for one particular detection chemistry, that is, AmpliFluor, Molecular Beacon, MGB, TaqMan, and SYBR-Green I. When possible, the assays were adapted to fast cycling mode. All assays displayed satisfactory performance parameters, although Molecular Beacon, MGB, and TaqMan chemistries were the most suitable for quantification purposes in both conventional and fast cycling modes. Keywords: Real-time PCR; genetically modified organism (GMO); detection chemistry; fast cycling mode; Mon810
ISSN:0021-8561
1520-5118
DOI:10.1021/jf063725g